Over the past four decades, tandem mass spectrometry has developed into an extraordinarily successful branch of mass spectrometry. A tandem mass spectrometer (MS/MS for short) first filters out a pre-selected ion species from a supply of an ion mixture, usually in the form of a continuous ion beam, fragments this ion species, and measures the spectrum of the fragment ions in a mass analyzer. The ions of the ion species selected are frequently called “parent ions”; the fragment ions are frequently called “daughter ions”.
The importance of tandem mass spectrometry lies in the fact that the acquisition of the fragment ion spectra provides insights into the structure of the parent ions selected, on the one hand and, on the other, enables certain identification of the type of the parent ions. In the biological sciences, it particularly enables sequences in biopolymers (or at least parts of these sequences and also modifications of these sequences) to be determined. It particularly makes it possible to determine amino acid sequences in proteins and peptides.
The importance of tandem mass spectrometry has further increased because the two ionization methods used almost exclusively for biomolecules, namely electrospray ionization (ESI) and matrix-assisted laser desorption and ionization (MALDI), are extraordinarily gentle (so-called “soft” ionization methods) and supply practically no fragment ions themselves, as was the case with the early ionization methods such as electron impact ionization. The soft ionization methods supply only so-called pseudo-molecular ions, usually protonated or deprotonated molecules, which only provide information about the mass of the molecule, but no further information concerning the identity and structure of the molecules. Further information is therefore required for structural analyses, even for certain identification of a substance, as is practically only provided by tandem mass spectrometry. Even if the aim is “only” a quantitative determination of a substance being sought which is actually known, certain identification, and therefore the use of tandem mass spectrometry, is indispensable in bio-analysis. The biological sciences therefore use tandem mass spectrometers in the majority of analyses.
For the information about the structures of the analyte substances obtained from their fragment ions with the aid of tandem mass spectrometry, it is favorable if the analyte ions to be fragmented are available in a doubly or multiply charged form. Some types of fragmentation can only be undertaken on multiply charged ions anyway. Electrospray ionization (ESI) in itself generates not only singly charged ions but also significant quantities of multiply charged ions; and it is noticeable that electrospray ionization therefore is gaining ground compared to other ionization methods. However, since electrospray ionization always requires a liquid phase, and since sample introduction via liquid phases is always quite slow, and also has a few other disadvantages, the increasing restriction of bio-analytical mass spectrometry to electrospray ionization is not entirely favorable. The often cited possibility of coupling with liquid chromatography or capillary electrophoresis makes the overall analysis slow, and there is only limited time to analyze the sample just supplied by a chromatographic peak.
The other important type of ionization for biomolecules is ionization by matrix-assisted laser desorption (MALDI). This ionizes the samples from the solid phase. Hundreds of samples can be applied on a sample support. Pipetting robots are available for this. The transport of the samples on the sample support into the laser focus takes only fractions of seconds, as much time as is ever needed is available for the analysis of this sample (until the sample is completely used up). MALDI is ideal for the identification of tryptically digested proteins which have been separated by 2D gel electrophoresis. MALDI analysis of peptides which have been separated by liquid chromatography is gaining ground (HPLC MALDI). A disadvantage of MALDI, however, is that it only ever supplies singly charged ions of the analyte substances.
The current methods for analyzing MALDI ions in time-of-flight mass spectrometers (MALDI TOF and MALDI TOF/TOF) have disadvantages, mainly in terms of an inadequate mass accuracy. The mass accuracy is still unsatisfactory even if each mass spectrum is subjected to a time-consuming mathematical recalibration using co-measured calibration substances. This inadequate mass accuracy stems from the fact that the MALDI process gives the ions an initial energy which differs from spectrum acquisition to spectrum acquisition and which, in a time-of-flight mass spectrometer operated with axial injection, leads to continuous shifts in the ion signals on the mass scale. This instability of the mass scaling is not known in other types of mass spectrometer, including time-of-flight mass spectrometers with orthogonal ion injection in particular.
We have already indicated that for tandem mass spectrometry, which is generally based on mass spectrometers with a stable mass scale, the fragmentation of the selected ions is a very important step. Over the past few years, it has been realized that a fragmentation which is rich in structural information very preferably starts with multiply charged ions; at least with doubly charged ions. This already applies for the oldest type of fragmentation, collisionally induced fragmentation (CID); newer types of fragmentation which are based on the transfer of electrons can only be initiated on ions which are at least doubly charged in any case. For tandem mass spectrometry with ion sources that supply only singly charged ions, a fundamental question is therefore how to produce ions which are at least doubly charged from the singly charged ions.
Until now, the electrospraying of dissolved bio-substances and the desorption of such substances by the impact of highly charged droplets (or clusters) have been the only ionization methods which lead to multiply charged bio-analyte ions. It seems as if there has to be a vaporization of the solvent from a highly charged droplet of the analyte solution in order to obtain multiply charged ions of the analyte substances. Other ionization methods, including the very interesting matrix-assisted laser desorption and ionization (MALDI), and also chemical ionization (CI) or photoionization (PI), lead only to singly charged ions.
For proteins and peptides it has now turned out that there are essentially two fundamentally different types of fragmentation of these biopolymers. These two types of fragmentation provide sets of information which are independent of each other (often termed “orthogonal” methods), and a comparison of the fragment ion spectra of the two types of fragmentation provides particularly valuable additional information. A tandem mass spectrometer which allows both types of fragmentation to be used on the same analyte ions is therefore particularly valuable.
The first type of fragmentation is a decomposition of the parent ions after they have collected sufficient internal energy from one or several energy absorption processes. The energy can be collected from a large number of moderate collisions (CID=collision induced decomposition), and also by absorbing a large number of infrared quanta (IRMPD=infrared multi photon decomposition). The internal energy here is distributed over all the internal oscillation systems of the parent ions, but the localization of the energy changes constantly because the oscillation systems are coupled and therefore continuously exchange energy among themselves. If, at a bond of the parent ion, a force finally occurs which exceeds the bonding force, then the parent ion breaks here into two fragments. Statistically, the cleavages only affect those bonds with low binding energies. In the case of proteins, this type of decomposition mainly leads to so-called b and y fragment ions. These b and y fragment ions are dissociated exactly at the peptide C—N bond locations connecting the amino group NH with the acid group COOH. For this first type of fragmentation it is therefore favorable to start with doubly charged ions because singly charged ions are difficult to fragment and form only very few types of fragment ions when they do fragment. The fragmentation of peptide ions here does not create any long signal sequences which mirror relatively long sections of the amino acid sequences. The fragment ion spectra therefore contain relatively little information if the starting point is singly charged ions.
One modification of this is the so-called high energy collisionally induced fragmentation (HE-CID). With collisions of kinetic energies in the region of a few kiloelectron-volts, a single collision is sufficient to lead to fragmentations. While it is possible here to start with singly charged ions, the fragment spectra generated in this way look more complicated than low energy CID fragment ion spectra because they contain more spontaneous fragmentations, for example the splitting off of side chains, and also more subsequent fragmentations (double and triple fragmentations with the appearance of so-called internal fragments) and therefore more fragment ion signals overall. While it is true that these fragment ion spectra have a high informational value thanks to the high energy collisions, they are difficult to interpret and tend therefore to be avoided. Basically, these high energy fragment ion spectra of proteins also contain predominantly b and y fragment ions.
The second, fundamentally different type of fragmentation is brought about by an electron transfer to multiply positively charged parent ions, thus neutralizing a proton; the decomposition is spontaneous and leads predominantly to so-called c and z fragment ions, broken at C—C bonds inside the amino acids, the c fragment ions generally being in the majority. This fragmentation process never splits off side chains like phosphorylations or even glycosilations. They provide fragment ions which are very easy to interpret and which are particularly suitable for the sequencing of unknown peptides and proteins (“de novo sequencing”), and for recognition and localization of modifications. This second type of fragmentation requires of necessity multiply charged ions, at least doubly charged ions, so that, after neutralization of a proton, a protonated ion still remains. The electron transfer can be brought about by direct capture of an electron (ECD=electron capture dissociation), by transfer of an electron of a negatively charged ion (ETD=electron transfer dissociation), or by the transfer of an electron from a highly excited atom to the parent ion (MAID=metastable atom induced dissociation).
It is particularly favorable to be able to apply both types of fragmentation to the same analyte ions since the comparison of the fragmentation spectra at constant mass differences makes it possible to immediately recognize which of the ion signals belong to b and c fragments, and which to y and z fragments. This makes the sequence very easy to unambiguously read off, which cannot be said for a single fragment ion spectrum from the mixing of two species of fragment ions.
The production of multiply negatively charged analyte ions from singly deprotonated analyte ions has already been described (“Increasing the Negative Charge of a Macroanion in the Gas Phase via Sequential Charge Reversion Reactions”, M. He and S. A. McLucky, Anal. Chem. 2004, 76, 4189-4192). The production here occurs in two stages, in which negative and positive ions with different proton affinities react with each other each time. This can only be undertaken in reaction cells which enable both negative and positive ions to be stored, for example in three-dimensional ion traps with ring and end cap electrodes. An analogous production of multiply positively charged ions has not yet been reported. The two-stage methods are also not particularly favorable for use in tandem mass spectrometers, however.
Whenever the term “mass of the ions” or simply “mass” is used here in connection with ions, it is always the “charge-related mass” m/z which is meant, i.e., the physical mass m of the ions divided by the dimensionless and absolute number z of the positive or negative elementary charges which this ion carries.
An “analysis” of an ion species or a substance is to be taken here as being both the determination of the quantity relative to other ion species or other substances (“quantitative analysis”), as well as the determination of the identity of the ion species or substance (“qualitative analysis”) via further measurements, for example from measurements of the internal structure of the ions, or even only the determination of the structure; in the case of biopolymers, the sequence of the modified or unmodified polymer building blocks of the ions of an ion species in general (“structural analysis”, “sequential analysis”, “modification analysis” etc.).